The disclosed invention relates to the field of imaging and analyzing the motion of motile organisms in general (e.g., cells, gametes, or single-celled organisms), and in particular to the field of imaging and analyzing sperm motility.
Analysis of sperm motility, i.e., the measurement of their ability to move properly, for the assessment of male reproductive health and the likelihood of successful outcomes in natural or artificial insemination has become a widely used tool in both agricultural and clinical diagnostics (R. Amann and D. Waberski (2014), “Computer-Assisted Sperm Analysis (CASA): Capabilities and Potential Developments”, Theriogenology 81:5-17; G. Di Caprio, et al. (2015), “Holographic Imaging of Unlabelled Sperm Cells for Semen Analysis: A Review”, J. Biophotonics 8(10):779-789). In humans, sperm concentration, morphology and motility measurements conducted as part of a semen analysis are used to assess male fertility. In agricultural settings, animal semen analysis is used in assessing the quality of semen samples, including previously frozen semen samples, for artificial insemination at stud farms and farm animal breeding facilities.
The rapid growth in the use of artificial insemination in the cattle industry starting in the late 1940s and early 1950s led to a need for objective methods to evaluate sperm quality (R. Amann and D. Waberski (2014)). Early approaches were based on microscopy-based observation, which through subsequent advancements in film-based or electronic imaging technologies, digital computing, and image processing software have led to the development of modern computer-assisted sperm analysis (CASA) systems. In a typical commercially-available CASA system, phase contrast microscope images of sperm (confined to motion in two-dimensions within a shallow sample chamber) are acquired using an image sensor which converts the images to digital data (at rates of 50 to 60 frames per second) that may be stored and manipulated using a computer and appropriate software. Image processing software algorithms perform edge detection and object (sperm cell) identification within each image frame, centroid calculations for each sperm cell detected within the field-of-view, tracking of centroids from one image frame to the next to identify trajectories or paths of motion, and estimation of the velocity or other motion parameters for each sperm cell detected within the field-of-view. CASA systems may provide a variety of in-plane motility data (for each individual sperm cell or for the population) such as straight-line velocities, curvilinear velocities, percentage of sperm exhibiting a velocity greater than a specified threshold value, and degree of linearity of motion (R. Amann and D. Waberski (2014); G. Di Caprio, et al. (2015)). Some CASA systems also provide sperm morphology analysis capabilities. A number of experimental and instrument design parameters may affect the accuracy and precision of CASA system output data, including sperm type, the type of extender or medium used for sample preparation, specimen chamber dimensions (in particular, chamber depth), the intensity of illumination, imaging hardware and software, instrument settings, technician training and skill level, etc.
The importance of CASA system data for assessing the product quality of semen marketed for artificial insemination of cattle, horses, or pigs is increasing (Amann & Waberski (2014)). Because most commercially-available CASA systems are quite large and expensive, there is a need for a field-use, portable CASA system capable of measuring the motion and/or morphology attributes of individual sperm. Such a system would be beneficial to veterinarians treating farm animal and race horse reproductive issues, veterinarians and technicians working at stud farms and farm animal breeding facilities that utilize artificial insemination techniques, and to physicians treating male reproductive problems in rural areas or smaller urban centers.